Chimeric cell-targeting pathogenic organism and method of therapeutic use

ABSTRACT

The invention chimeric organism comprises a chimeric surface integrin-like fusion protein in which the I domain has been replaced by an antibody fragment that binds a disease-associated antigen on a cell. Binding of the antibody fragment to the disease-associated antigen triggers virulent transformation of the chimeric pathogenic organism so as to cause the organism to infiltrate the target cell with specificity. Preferably, the chimeric organism is a chimeric pathogenic  C. albicans  having an INT1 fusion protein in which the I domain is replaced by an antibody fragment, preferably a single chain antibody, and in which expression of an iron transporter gene necessary for infiltration of a target cell is triggered under the control of a EFG1p response element. Binding of the antibody to the disease-associated antigen causes filamentous transformation in the chimeric pathogenic  C. albicans  and specific infiltration of target cells. The invention chimeric pathogenic organisms are used in therapeutic methods to specifically infiltrate and destroy diseased cells to which the antibody fragment binds while remaining non-pathogenic to normal cells.

FIELD OF THE INVENTION

[0001] This invention relates to treatment of diseases characterized byproduction of cell surface markers using antibody-targeted compositions.More particularly, this invention relates to chimeric organisms thatexpress an antibody fragment and to the use of such chimeric organismsin treatment of diseases characterized by production of cell surfacemarkers.

BACKGROUND OF THE INVENTION

[0002] Many recent gene therapy approaches have exploited thespecificity of antibody binding to target cancer cell lines in order todeliver either drugs or immune responses to an actual tumor location.Most cancer cell lines misregulate cell surface proteins andpolysaccharides, and are thus easily distinguished from normal somalcells by antibodies (R. E. Hawkins et al., Gene Therapy (1998),5:1581-1583). It is apparent that established carcinomas havesuccessfully avoided activating the immune response within their hosts.Direct attempts to rectify this by recruiting the body's humoral immuneresponse to tumors by injection of murine derived antibodies canunfortunately cause serious and even life threatening human anti-mouseresponses (R. K. Jain et al., J Natl. Cancer Inst. (1989) 81:570-576 andD. Colcher et al., J. Nat. Cancer Inst. (1990) 82:1191-1197). Inaddition, the overall penetration of antibodies into tumors is limiteddue to the high molecular weights of these molecules (K. A. Chester etal., Adv. Drug Delivery Rev. (1996) 22:303-313).

[0003] In an attempt to limit both the size of the antibody and themouse-character of the antibody, single chain antibodies (scFvs) thatencapsulate the binding features of the Fv region of the antibodywithout the bulk of the native antibody sequence in the c1, c2, and c3domains have been developed. One methodology to generate scFvs involvestethering the antigen binding domains of V_(H) and V_(L) together usinga short flexible peptide linker (R. E. Bird et al., Science (1988)242:423-426). Another approach involves the generation de novo ofmolecular diversity, instead of generating monoclonal antibodies inmice. By using combinatorial antibody libraries on the surface offilamentous bacteriophage screened against immobilized antigen, a singlepolypeptide chain that is amenable to fusion with other proteins can begenerated (J. S. Huston et al., Proc. Natl. Acad. Sci. USA (1988)85:5879-5883; J. McCafferty, Nature (1990) 348:552-554; R. H. J. Begentet al., Nature Med. (1996) 2:979-984, reviewed in K. A. Chester et al.,Adv. Drug Delivery Rev. (1996) 22, 303-313). The scFvs obtained byeither methodology above show better tumor penetration, but therapeuticapplication is still in early stages (G. Reitmuller et al., Lancet(1994) 343: 1177-1183). However, fusions between imaging agents andscFvs have found wide acceptance and extensive application in tumorimaging and radiochemotherapeutic delivery (see J. Bhatia et al., Cancer(1999) 85:571-577 and A. M. Wu et al., Tumor Targeting (1999) 4:47-58and references therein).

[0004] Antibody recognition has also been used to target cancer cells byincorporation of an scFv into the envelope protein of a retrovirus (S.J. Russell et al., Nuc. Acids Res. (1993) 21:1081-1085 and F. Martin etal., Human Gene Therapy (1998) 9:737-746). This targeting is modest, butoffers some promise, as has been demonstrated for certain types ofmelanoma (Martin 1998). In addition, adenovirus infection has been usedto allow the transient expression of tumor-targeting scFv fusionproteins in whole organisms with moderate success (H. A. Whittington etal., Gene Therapy (1998) 5:770-777). Unfortunately, low survivability ofadenoviruses carrying antibody generating expression vectors limitstheir impact.

[0005] The most promising therapeutic techniques relying on thespecificity of antibody binding focus on engineering T-cells thatexpress antibody fragments fused to surface proteins, and are thusdirected to tumor surfaces (recent work reviewed in F. Paillard, HumanGene Therapy (1999) 10:151-153). Some of these T-cells are at present inclinical trials. Strategies used to date, however, have drawbacks,including limited efficacy against established tumors, thoughdemonstrating some slowing of tumor metastasis (R. P. McGuinness et al,Human Gene Therapy (1999) 10:165-173). Limited effectiveness againstestablished tumors may be due to the inability of the T-cells topenetrate solid cell masses (Paillard 1999). True protection againstestablishment of invasive carcinoma was obtained only by coinjection ofmodified T-cells with the tumorogenic line. In clinical applications,this may permit stabilization and localization of established tumors,but not reductive treatment. Another potential problem is that suicidesignals T-cells use to induce apoptosis, like tumor necrosis factor I,are often not functional against carcinomas. Even when they areeffective, successful cancer cell lines will rapidly adapt to apoptoticsignals, and have even been known to induce apoptosis in attackingT-cells (K. Shiraki, Proc. Natl. Acad. Sci. USA (1996) 94:6420-6425). Inaddition, T-cells bearing these chimeras are assembled separately foreach patient ex vivo due to possible MHC incompatibilities that couldresult in serious allergic reactions were T-cells from other humansintroduced therapeutically.

[0006]Candida albicans is the most commonly isolated invasive fungalpathogen in humans. This organism is representative of several thatswitch between two major classes of morphology. The first morphology isthe ellipsoid blastospore. Like most yeast, C. albicans assumes thisarchitecture when growing non-pathogenically. Upon binding of C.albicans to mammalian tissues (i.e. via the I domain of the INT-1protein), the cell morphology switches to various filamentous forms,including germ tubes and hyphae, that are capable of aggressivelyinvading host tissue (reviewed by R. A. Calderone, Microbol. Rev. (1991)55, 1-20). Systemic infection of a vulnerable host by C. albicansresults in high levels of mortality. For example, more than 30% ofimmunocompromised HIV patients are systemically infected despiteappropriate treatment regimes. In addition, C. albicans infectioncommonly leads to death in premature infants, diabetics, and surgicalpatients. To date, the ability of this pathogenic organism to infectcells when the cell morphology switches to a filamentous form has notbeen utilized for therapeutic purposes, such as in cancer therapy.

[0007] Thus, the need exists in the art for new and better compositionsand methods of their use for treating various types of cancers and otherdiseases associated with production of an abnormal protein.

SUMMARY OF THE INVENTION

[0008] The present invention overcomes these and other problems in theart by providing chimeric organisms having a chimeric surfaceintegrin-like protein in which the I domain has been replaced by anantibody fragment that binds a disease-associated antigen on a cell.Binding of the antibody fragment to the disease-associated antigen onthe cell triggers virulent transformation of the chimeric pathogenicorganism and allows the organism to infect the cell.

[0009] In one embodiment according to the present invention, there areprovided chimeric pathogenic C. albicans modified to contain anintegrin1 (INT1) fusion protein in which the I domain is replaced by anantibody fragment that binds to a disease-associated antigen on adiseased cell. The chimeric C. albicans further contains a disabledwild-type high affininity iron transporter (CAFTR) gene, and a DNAconstruct comprising a wild-type CAFTR gene under the control of anenhanced filamentous growth protein (EFG1p) response element, whereinbinding of the antibody to the disease-associated antigen triggersexpression of the CAFTR gene in the DNA construct and filamentoustransformation in the chimeric pathogenic C. albicans.

[0010] In another embodiment according to the present invention, thereare provided methods for treating a disease associated with the presenceof cells having a disease-associated surface antigen in a subject inneed thereof by administering to the subject a therapeutically effectiveamount of an invention chimeric pathogenic organism so as to causebinding of the antibody fragment to the disease-associated antigen onthe cells, thereby treating the disease by triggering infiltration ofthe chimeric pathogenic C. Albicans into the cells without substantialdamage to healthy cells.

[0011] In yet another embodiment, the present invention provides methodsfor generating a chimeric therapeutic organism from a pathogenicorganism that possesses in the wild-type an integrin-like protein withan I domain. In the invention methods, the I domain in the integrin-likeprotein of the pathogenic organism is replaced with an antibody fragmentthat binds to a disease-associated antigen on a diseased cell. In thechimeric therapeutic organism, virulent transformation occurs uponbinding of the antibody fragment to the disease-associated antigen onthe cell.

A BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1A-W show the nucleotide sequence of the gene that encodesthe integrin-like INT1 protein of C. albicans (GenBank Accession#U35070) (SEQ ID NO:1)

[0013]FIG. 2 shows the nucleotide sequences of seven primers used inconstruction of the chimeric C. albicans of Example 1 (SEQ ID NOS:2through 8, respectively).

[0014]FIG. 3 is a schematic drawing showing human integrin structure(adapted from M. J. Humphries, Biochem. Soc. Trans. (2000) 28:311-340).

[0015]FIG. 4 is a schematic drawing showing two pathways by which hyphaldevelopment in yeast is regulated.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides chimeric pathogenic organismsderived from wild type organisms wherein virulent transformation of theorganism is controlled in the wild-type organism by binding of the Idomain of a surface integrin-like protein to a cell. The inventionchimeric organism comprises a chimeric surface integrin-like protein inwhich the I domain is replaced by an antibody fragment that binds adisease-associated antigen on a cell. Binding of the antibody fragmentto the disease-associated antigen triggers virulent transformation ofthe chimeric pathogenic organism so as to cause the organism toinfiltrate the cell. Virulent invasion of the cell by the chimericpathogen inhibits growth of the diseased cell.

[0017] The invention pathogenic chimeric organism represents a newapproach to employing otherwise pathogenic organisms to assist indisease treatment. Although the present invention is described forillustrative purposes with reference to a reingeneered C albicans,suitable pathogenic organisms in addition to C. albicans that can beengineered according to the methods disclosed herein are pathogenicorganisms that become virulent (e.g., switch to a filamentous invasiveform) upon binding of its integrin-like surface protein (i.e., acell-cell communication protein) to a target on another cell and inwhich the binding domain of the surface protein can be replaced with aantibody fragment that binds to a desired target cell associated with adisease state. Preferably the chimeric pathogen also is relativelyharmless to mammalian cells until binding of the antibody fragmentcontained in its surface protein.

[0018] In higher eukaryotes, integrins are one of the most importantclasses of surface proteins responsible for intercellular communication(reviewed in F. G. Giancotti Science (1999) 285:1028-1032 and M. J.Humphries, Biochem. Soc. Trans. (2000) 28:311-340). Generally, integrinsare heterodimers, each subunit of which consists of a cytosolic domainwith one tyrosine used as a kinase regulatory site, a transmembranedomain, and four EFG-like repeats. As used herein, the term“integrin-like protein” refers to a cell-cell communicationtransmembrane protein that contains one or more of the above features.

[0019] There are various other domains on the integrin proteins,including metal binding MIDAS loops and β propeller domains. Notably, innine of the fifteen human integrin I subunits, there is a protrudingregion known alternately as the IA, or the I domain, which appears toregulate integrin targeting. This suggests that the absence or presenceof the I domain has little, if any, effect on the integrin's ability totransduce signals, but instead regulates which signals are transduced.The I domain is the only region whose structure has been solvedcrystallographically (both bound to its target proteins and unbound).Based on these studies, it is believed that the I domain alone is indeedsufficient for binding to collagen (J. Emsley Cell (2000) 101:47-56).

[0020] In the invention chimeric organism, the endogenous binding regionof the surface integrin-like protein, which nonspecifically targetscells (e.g., those containing fibrinogen), is replaced with an antibodyfragment, such as a single chain antibody. As a result, rather thannonspecifically binding to any cell containing a binding site for theendogenous binding region, the invention chimeric pathogen binds withspecificity to cells that express the target antigen. Binding of thechimeric pathogen to a cell containing an epitope for the antibodyfragment triggers virulent invasion of the disease-associated cell.Other cells (e.g., healthy cells) are not bound by the chimericorganism. As a result, pathogenic infiltration of non-targeted cellsdoes not take place.

[0021] In one embodiment, the invention provides a chimeric pathogenicC. albicans comprising an INT1 fusion protein in which the I domain isreplaced by an antibody fragment that binds to a disease-associatedantigen on a cell. Preferably, the INT1 protein in the inventionpathogenic organism is a fusion protein in which a single chain antibodyreplaces the native I domain. The nucleotide sequence encoding INT1 isshown in FIGS. 1A-W (SEQ ID NO:1 herein). Construction of such achimeric INT1 is described in Example 1 below.

[0022] As used herein, the term “disease-associated antigen” meanseither that the antigen is not expressed in normal, healthy cells, orthat the antigen is expressed in abnormal quantity in diseased cells.Existence of disease-associated antigen on cells greatly increases theamount of the chimeric pathogen that attacks such disease-associatedcells.

[0023] Preferably the antibody fragment is a single chain antibody(scFv),), but Fv and CDR fragments can also be used. The antibodyfragment is preferably incorporated into the surface integrin-likeprotein to form a fusion protein.

[0024] In a preferred embodiment, the chimeric pathogen is a chimericCandida albicans, the most common fungal pathogen of humans. Inimmunocompromised individuals, C. albicans is a dangerous, and sometimeslethal pathogen. The primary protein responsible for C. albicanstargeting is an integrin-like transmembrane protein known as INT1. INT1contains an integrin-like domain (known as the I domain), which is theputative targeting region of this protein. FIGS. 1A-W show thenucleotide sequence of the gene that encodes the integrin-like INT 1protein of C. Albicans (GenBank Accession #U35070) (SEQ ID NO:1).

[0025] The preferred antibody fragment for incorporation into the INT1protein as a fusion protein is a single chain antibody (scFv), but Fvand CDR fragments can also be used. Thus, in this embodiment, the powerand specificity of scFv antibody fragments, which now exist against aconsiderable number of cell surface targets in cancer cell lines, iscombined with the ability to not only bind to the cancer cell mass, butto invade and destroy tumors aggressively and selectively in a mannerindependent and complementary to the body's own defenses.

[0026] The present invention exploits the mechanism involved in thefilamentous transformation of C. albicans and similar pathogenicorganisms, which is controlled by a regulatory system similar to thatused by Saccharomyces cerevisiae to alter its own morphology undernitrogen starvation conditions and agar invasive conditions. Theevolutionary conservation of this pathway has greatly facilitateddeconvoluting the biochemistry involved, and recent research hasresulted in a better understanding of proteins responsible for C.albicans intercellular binding and pathogenicity. In particular, INT1 isthe previously known but unidentified surface protein that is stronglycrossreactive with antibodies for certain leukocyte integrins, andappears to be the primary protein responsible for attaching to targetcells (C. A. Gale et al., Science (1998) 279:1355-1358). INT1 is atransmembrane surface protein isolated by cDNA screening of the C.albicans genome by oligonucleotide probes derived from the conservedregion of human integrins (C. Gale et al., Proc. Natl. Acad. Sci. USA(1996) 93:357-361). Over the last fifteen years, many reports haveappeared in the literature describing surface proteins that are relatedto I-subunits of the leukocyte integrins IM/θ2 (Mac-1; CD11b/CD18) andIX/θ2 (p150,95; CD11c/CD18) (C. M. Bendel, J. Clin Invest. (1993)92:1840-1849 and references therein). Many monoclonal antibodies thatrecognize epitopes of these leukocyte surface proteins cross react toblastospores and germ tubes of C. albicans, sometimes with the sameaffinity as to the original human targets.

[0027] Upon ligand binding, INT1 signals cell morphology changes thatinduce hyphae growth. This signaling pathway appears to be largelyindependent of the mating factor MAPK pathway, which is most commonlyassociated with morphologic changes. Unlike the pathway that istriggered by ligand binding, the MAPK pathway is triggered byenvironmental stimulation, such as changes in pH, temperature, matingsignaling or nutrient availability, and terminates in transcriptionfactor STE12 in S. cerevisiae (Gale 1996; C. J. Gimeno et al., Cell(1992) 68, 1077-1090 1992 and R. L. Roberts et al., Genes Dev. (1994) 8,2974-2985) (FIG. 4).

[0028] The second pathway to hyphal morphology, which is less wellcharacterized, depends on direct stimulation by serum. Addition ofmammalian serum to an otherwise spheroplast culture of C. albicansinduces hyphae growth, even when the signaling cascade terminating inSTE12 (briefly described above) is completely knocked out (Lo 1997).INT1 instead communicates morphology changes via a second, STE12independent, pathway that appears to have as an intermediary proteinAsh1. Ash 1 is a daughter cell-specific protein that helps regulatefilamentous growth, and may interact with STE20 (S. Chandarlapaty etal., Mol. Cell Biol. (1998) 18, 2884-2891). This pathway terminates atthe transcriptional level in a bHLH class protein known as PHD1 recentlyisolated in S. cerevisiae. The homologous protein in C. albicans isknown as EFG1. The sequence for EFG1 is fully described in W. R. Stoldtet al., EMBO Journal (1997) 16, 1982-1991, which is incorporated hereinby reference in its entirety.

[0029] For both S. cerevisiae and C albicans, overexpression of theirrespective analog is sufficient to induce hyphae growth (Stoldt 1997).Importantly, elimination of filamentous growth can only be achievedafter disabling both of these two pathways. This observation also rulesout a third pathway for signaling.

[0030] In construction of the illustrative C. albicans chimericpathogen, the integrin homolog INT1 was isolated using conservedtransmembrane sequences from mammalian integrins to clone the cDNA copyof the gene in C. albicans. Removal of the INT1 protein reduces specificadhesion of C. albicans to HeLa cells by 39%. Therefore, though INT1 isa critical protein for binding to a target, other proteins as well mustserve to help mediate this interaction (vide infra), though theseproteins have yet to be identified. Still, previous research appears tobe consistent with cell surface binding by a single, integrin-likeprotein of approximate MW of 165 kDa. Interestingly, transgenicexperiments where INT1 was overexpressed on the surface of Saccharomycescerevisiae, a nonadhesive and nonpathogenic species, caused strongadhesion to mammalian cells. Thus, INT1 alone is sufficient for targetbinding.

[0031] Deletion of the INT1 gene cripples filamentous growth of Candida,though not entirely eliminating it. It has been shown that invasivegrowth of this type is necessary for parasitic microorganisms tosuccessfully invade host tissues (W-S Lo et al., Mol. Biol. Cell (1998)9:161-171). In vivo testing of the pathogenicity of an INT1 ofpathogenicity of the INT1 deletion strain of C. albicans on mice wasconducted and showed a dramatic reduction in mouse lethality compared towild-type strains (Gale 1996).

[0032] The similarity of INT1 in C. albicans to mammalian integrins isnot limited to antibody cross reactivity and sequence similarity in thetransmembrane region. Notably, INT1 also appears to contain numerousmotifs similar by homology to mammalian integrin motifs. These include(1) two FE-hand divalent cation binding sites that likely mediate targetbinding; (2) a single cytosolic tyrosine for kinase signaling; and, mostimportantly, (3) a region that appears to be homologous to the I domainof integrins. Similar to the higher mammalian IM and IX that recognizeiC3b and fibrinogen, the I domain like region in C. albicans INT1 isgenerally thought to be the binding site that targets iC3b. This isfurther supported by its ˜25% sequence identity with the fibrogenbinding domain of Staphylococcus aureus.

[0033] In the illustrative preferred embodiment of the inventionchimeric pathogen, the I domain of the wild-type INT1 protein, whichnonspecifically targets fibrinogen, is replaced with an scFv thattargets cancer cells. Many scFvs already have been developed that bindto a wide variety of tumor cells for therapeutic applications. Suchstudies take advantage of the severe misregulation of surface proteinpopulations in tumors by utilizing scFvs that bind epitopes found insuch surface proteins. For example, therapeutic applications involvingT-cell, viral, and/or drug targeting has already been proven in vivousing scFvs shown in Table 1 below. TABLE 1 CANCER LINE AND ANTIBODYANTIGEN LOCATION REFERENCES OTHER CC49 TAG-72 Adenocarcinoma McGuiness1999 (colon, ovarian, breast) Shu 1993 FRP5 ERBB2 Breast, ovarianKashmiri 1995 Moritz 1994 Previously used to Harwerth 1992 constructcytotoxic Hynes 1993 C-lymphocytes. Also used to direct virus targeting(Galmiche 1997) GA733.2 EGP-2 Various Ren-Heidenreich 2000 HMN-14 CEAColorectal, breast, Nolan 1999 Previously used to pancreas, otherconstruct killer T-cells VFF17 CD44 Cervical cancer, lymph Dall 1997metastases Hekele 1996 MOV19 I-FR Nonmucinous ovarian Melani 1998carcinoma 7.16.4 Neu Breast Katsumata 1995 Antigen (neu) is sameStankovski 1993 as ERBB2, and is Disis 1997 protein bound by Herceptin.MLuC1 L(Y) TAA Various Mezzanzanica 1998 Targets misregulatedcarbohydrates. Lewis (Y) tumor associated antigen

[0034] By replacing the I domain in the integrin-like surface proteinwith a scFv that binds to a disease-associated tumor cell, the inventionchimeric pathogenic organisms are engineered to take advantage of theseverely misregulated production of surface protein populations intumors. In the present invention, the antibody fragment, preferably as ascFv, is incorporated into the position of the native binding domain ofan integrin-like protein (i.e., the creation of a fusion protein thatcontains the scFv incorporated in the place of the I domain in thewild-type pathogenic organism). Many antibody fragments have alreadybeen tested for selective binding to a known tumor-associated antigen,for example, as shown in Table 1. Representative non-limiting examplesof tumor associated antigens to which scFvs of the invention chimericpathogens bind include GAG-72, ERBB2, EGP-2, CEA, CD44, I-FR, neu, theLewis (Y) tumor associated antigen, and the like.

[0035] As used herein, the terms “disease- or tumor-associated antigen”and “disease- or tumor-associated epitope” encompass antigens andepitopes, respectively, found in surface proteins produced in largeamounts in various types of tumors as well as various types of markerproteins (and the epitopes contained therein) that are found associatedwith tumor cells and not found associated with normal cells.Representative non-limiting examples of tumors having associatedantigens to which antibody fragments (e.g., scFvs) of the inventionchimeric pathogens bind includes adenocarcinoma of colon, ovary orbreast; cervical cancer, nonmucinous ovarian carcinoma; breast, ovarian,colorectal, and pancreatic cancer, and the like. Invention chimericpathogenic organisms are incapable of infiltrating a cell in the subjectuntil the antibody fragment in the chimeric integrin-like protein bindsto its target epitope, triggering a virulent transformation of thechimeric pathogenic organism. Therefore, the invention chimericpathogenic organisms are substantially incapable of pathogenic activity,such as infiltration, of cells other than their target cells (e.g.,cancer cells).

[0036] Preferably, the antibody fragment is a scFv and is introduced inthe place of the I domain of INT-1 in C. albicans. Once engineered toreplace the wild-type binding domain of the INT1 protein with an antigenbinding region (e.g. scFv) from cancer-specific antibodies, theinvention mutant C. albicans strain will specifically bind to a cancerline dictated by the targeting of the scFv-INT1 fusion protein.

[0037] Optionally, in order to direct pathogenicity specifically to thetarget cell (e.g., a carcinoma cell) a gene in the pathogenic organismfrom which the chimeric organism is derived that is required forinvasive growth is disabled or removed and a DNA construct comprising areengineered copy of the gene necessary for invasive growth isintroduced into the chimeric organism under the regulatory control of atranscription factor that regulates filamentous transformation of theorganism. However, the gene removed should be one that does notsignificantly affect vegetative growth of the organism so that largequantities of this chimeric organism can be produced using standardculture techniques.

[0038] For example, in C albicans, the wild-type gene is placed underthe control of a EFG1p response element. While the CaFTR1 gene iscurrently preferred for reengineering in C. albicans, those of skill inthe art can readily substitute for reengineering (i.e., in the place ofthe CaFTR1 gene) another gene from the pathogenic organism that isessential or preferred for pathogenic invasion.

[0039] Preferably, in the invention chimeric C. albicans, the wild-typeCAFTR gene is either disabled or removed and a DNA construct comprisinga wild-type CAFTR gene under the control of a EFG1p response element isintroduced. Overexpression of EFG1 in C. albicans leads to enhancedfilamentous growth in liquid and on solid media. Overexpression of EFG1by a PCK1p-EFG1 fusion is described by A. Sonneborn, Infect Immun (1999)67:9:4655-60, which is incorporated herein by reference in its entirety(See also, V. R. Stoldt et al., EMBO J (1997) 16:8 1982-91). Thenucleotide sequence for the CaFTR1 gene of C. albicans is found at NCBIGenBank Number AF195775.

[0040] CaFTR1 extracts iron from mammalian tissues that withhold metalsfrom microbial predators as a defense mechanism (D. M. DeSilva et al.,Physiol Rev. (1996) 76, 31-47 and H. Gunshin et al., Nature (1997) 388,482-488). Removal of the native CaFTR1 completely abrogatespathogenicity. Mice injected with a mutant C. albicans having a disabledCaFTR1 gene survive entirely; while those injected with an equal amountof wild-type C. albicans do not. Under circumstances of normalunicellular growth in an abundance of iron, though, CaFTR1 is not anessential gene. In conditions where iron is in limited quantities, forinstance during circulation through a host designed to have limitingnutrient levels, this gene is highly upregulated. Removal of the CaFTR1gene only causes a growth (and thus invasion) deficiency whenpathogenesis is initiated. This protein is normally regulated entirelyindependently from the morphology signaling pathway, and itsconcentration is dependent only on the heavy metals detected in theenvironment. By placing this protein under the transcriptional controlof the cell morphology pathway initiated by INT1, as described herein,the pathogenicity of the overall assembly can be tightly restricted toscFv-INT1 targeted cells.

[0041] In this preferred embodiment of the invention chimeric pathogenicorganism, binding of the antibody to the disease-associated antigentriggers both expression of the CAFTR gene in the DNA construct andfilamentous transformation of the chimeric pathogenic C. albicans. Sinceexpression of INT1 in wild-type C. albicans is activated by INT1 bindingto other cells, placing expression of the C. albicans iron transporterunder control of the Efg1 expression system ensures that both thepathogenicity and the binding of the invention chimeric organism isdirected specifically to target cells. If the antibody fragmentincorporated in the place of the INT1 binding domains is specific for atumor cell, the pathogenicity of the C. albicans cell line is directedspecifically towards target cancerous cells, and nonspecific toxicity isinhibited. In other words, the virulence of the engineered strain of C.albicans will only be activated once scFv-INT1 binds to its targetantigen on the surface of the carcinoma.

[0042] The gene that triggers filamentous growth can be disabled in theinvention chimeric organism using any method known in the art, forexample, by disruption of the gene at both diploid loci using standardtechniques. The native gene can be reintroduced under the control of aresponse element (e.g., a transcription factor) that regulatesfilamentous transformation of the organism using known techniques, suchas by use of homologous recombination (as described in Example 1herein). This regulatory reassignment of the gene that triggerstransformation tightly limits the pathogenicity dependent on thisprotein to the specified target.

[0043] In yet another embodiment, the present invention provides methodsfor treating a disease associated with the presence of cells having adisease-associated surface antigen in a subject in need thereof. Theinvention method includes administering to the subject, atherapeutically effective amount of an invention chimeric pathogenicorganism so as to cause binding of the antibody fragment to thedisease-associated antigen on the cell, thereby specifically treatingthe disease by triggering infiltration of the chimeric pathogenicorganism into the cells without substantial damage to healthy cells. Theinvention method may further include administering to the subject animmunosuppressive agent to inhibit the subject's immune system fromdestroying the chimeric pathogen prior to achieving a therapeuticeffect. Representative immunosuppressive agents useful in the practiceof the invention methods include such agents as cyclosporin A, OKT3,FK506, mycophenolate mofetil (MMF), azathioprine, corticosteroids (suchas prednisone), antilymphocyte globulin, antithymocyte globulin, and thelike. In a preferred embodiment of the invention methods, the inventionchimeric pathogen is used to target and attack tumor cells.

[0044] In invention therapeutic methods, the chimeric pathogenicorganisms are used to infiltrate and destroy both ex vivo cell lines(e.g., tumor cell lines), as well as in vivo murine models of humancarcinomas, and the like. The invention pathogenic organisms also havespecific utility as research reagents for the testing of therapeuticcompositions. For example, the invention chimeric organisms can be usedto compare the therapeutic effect against a particular cell line ofvarious antibody fragments engineered into the surface integrin-likeprotein. Binding of invention organisms to these ex vivo and in vivomodels can be tested for efficacy using known assays (e.g., mouse tumormodels) to determine binding of the antibody fragment (e.g., singlechain antibody) to the target antigen on a disease-associated cell.

[0045] The chimeric pathogens used in practice of the invention methodcan be administered for therapeutic purposes, such as treatment oftumor, by any route known to those of skill in the art, such asintraarticularly, intracisternally, intraocularly, intraventricularly,intrathecally, intravenously, intramuscularly, intraperitoneally,intradermally, intratracheally, intracavitarily, and the like, as wellas by any combination of any two or more thereof.

[0046] The most suitable route for administration will vary dependingupon the disease state to be treated, or the location of the suspectedcondition or tumor to be treated. For example, for treatment ofinflammatory conditions and various tumors, local administration,including administration by injection directly into the body partcontaining the tumor provides the advantage that the chimeric pathogencan be administered in a high concentration without risk of thecomplications that may accompany systemic administration thereof.

[0047] The chimeric pathogen is administered in “a therapeuticallyeffective amount.” An effective amount is the quantity of a chimericpathogen necessary to aid in treatment, inhibition or destruction ofdiseased tissue (e.g. tumor) under treatment in a subject. A “subject”as the term is used herein is contemplated to include any mammal, suchas a domesticated pet, farm animal, or zoo animal, but preferably is ahuman. Amounts effective for therapeutic use will, of course, depend onsuch factors as the size and location of the body part to be treated,the affinity of the antibody fragment for the target antigen, the typeof target tissue, as well as the route of administration. Localadministration of the targeting construct will typically require asmaller dosage than any mode of systemic administration, although thelocal concentration of the chimeric pathogen may, in some cases, behigher following local administration than can be achieved with safetyupon systemic administration.

[0048] The invention composition can also be formulated as a sterileinjectable suspension according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1-4, butanediol. Sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides, fatty acids (including oleic acid), naturally occurringvegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil,etc., or synthetic fatty vehicles like ethyl oleate, or the like.Buffers, preservatives, antioxidants, and the like, can be incorporatedas required, or, alternatively, can comprise the formulation.

[0049] Preferably the antibody fragment is a scFv incorporated into thechimeric surface protein of the pathogen as a targeting device and isnot relied upon as the toxic agent. Rather, it is the pathogenicorganism itself that invades and destroys the target cells in accordancewith the present invention. A single chain antibody (scFv) is agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are known in the art. (Seefor example, Harlow & Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference). Asused in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as specific charge characteristics.

[0050] Fv fragments comprise an association of V_(H) and V_(L) chains.This association may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chainscan be linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. See, e.g., Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993. Preferably,the Fv fragments comprise V_(H) and V_(L) chains connected by a peptidelinker. These single-chain antigen binding proteins (scFv) are preparedby constructing a structural gene comprising DNA sequences encoding theV_(H) and V_(L) domains connected by an oligonucleotide. The structuralgene is inserted into an expression vector, which is subsequentlyintroduced into a host cell such as E. coli. The recombinant host cellssynthesize a single polypeptide chain with a linker peptide bridging thetwo V domains. Methods for producing scFvs are described, for example,by Whitlow et al., Methods: a Companion to Methods in Enzymology, 2: 97,1991; Bird et al., Science 242:423-426, 1988; Pack et al.,Bio/Technology 11:1271-77, 1993; Sandhu, supra, and Ladner et al., U.S.Pat. No. 4,946,778, which is hereby incorporated by reference in itsentirety.

[0051] Another form of an antibody fragment suitable for incorporationas a fusion protein in invention chimeric pathogenic organisms is apeptide coding for a single complementarity-determining region (CDR).CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: a Companion to Methods inEnzymology, 2: 106, 1991.

[0052] Antibodies that bind to a tumor cell or other disease-associatedantigen can be prepared using an intact polypeptide or biologicallyfunctional fragment containing small peptides of interest as theimmunizing antigen. The polypeptide or a peptide used to immunize ananimal (derived, for example, from translated cDNA or chemicalsynthesis) can be conjugated to a carrier protein, if desired. Commonlyused carriers that are chemically coupled to the peptide include keyholelimpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), andtetanus toxoid, and the like. The coupled peptide is then used toimmunize the animal (e.g., a mouse, a rat, or a rabbit).

[0053] The preparation of such monoclonal antibodies is conventional.See, for example, Kohler & Milstein, Nature 256:495, 1975; Coligan etal., sections 2.5.1-2.6.7; and Harlow et al., in: Antibodies: aLaboratory Manual, page 726 (Cold Spring Harbor Pub., 1988), which arehereby incorporated by reference. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes et al., Purification of Immunoglobulin G (IgG), in: Methods inMolecular Biology, Vol. 10, pages 79-104 (Humana Press, 1992).

[0054] Antibodies of the present invention may also be derived fromsubhuman primate antibodies. General techniques for raisingtherapeutically useful antibodies in baboons can be found, for example,in Goldenberg et al., International Patent Publication WO 91/11465(1991) and Losman et al., 1990, Int. J. Cancer 46:310, which are herebyincorporated by reference. Alternatively, a therapeutically usefulantibody may be derived from a “humanized” monoclonal antibody.Humanized monoclonal antibodies are produced by transferring mousecomplementarity determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and thensubstituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833,1989,which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al.,Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993, which arehereby incorporated by reference.

[0055] It is also possible to use anti-idiotype technology to producemonoclonal antibodies, which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hyper variable region that is the“image” of the epitope bound by the first monoclonal antibody.

[0056] The assembly, selection, and integration of these chimericscFv-INT1 products are conducted using standard molecular biology, forexample as is described in Example 1 herein. The proper assembly of theinvention chimeric scFv-INT-1 protein and adhesion to the epitopes intarget cell lines, e.g., tumor cell lines, can be tested by introductionof the chimeric assembly into S. cerevisiae, preferably under thecontrol of a promoter, such as the actin promoter, that is constantlyactivated in such yeast cell lines. Yeast cells (e.g., Saccharomycescerevisiae) possess an efficient and precise system for geneticrecombination. The natural process of homologous recombination dependson a system of enzymes that search for regions of sequence homologybetween two DNA molecules (which may be entire chromosomes). Oncehomology is found, an exchange of information is possible.

[0057] Plasmids or other vectors carrying recombinant-DNA (r-DNA) cloneswhich contain naturally-occurring yeast sequences and which areintroduced into cells by standard transformation methods are capable ofstably integrating into the yeast genome at sites of homology. Theefficiency of this process can be increased by up to a thousand-fold byintroducing a double-strand break within a DNA sequence on the incomingDNA molecule that is homologous to a sequence resident in the yeastcell. The cloned yeast DNA on the transforming vector is referred toherein as the targeting sequence, and the site of integration isreferred to herein as the target site.

[0058] In one process described in U.S. Pat. No. 5,783,385 to Treco , etal., which is incorporated herein by reference in its entirety, atargeting DNA molecule, e.g., a bacterial plasmid, which isnon-replicating in yeast is introduced into the population of host yeastcells containing the r-DNA. The bacterial plasmid has a selectablemarker gene that functions in yeast and a first targeting DNA sequencewhich is homologous in part to a second target r-DNA clone sequence.Preferentially, the targeting plasmid is cut with a restrictionendonuclease that introduces a double-strand break within the targetingsequence, thereby linearizing the bacterial plasmid and providing DNAends which are recombinogenic to stimulate the process of homologousrecombination with host yeast sequences. Because the plasmid isnon-replicating in yeast, stable transformation with the selectablemarker can only proceed by homologous recombination. The efficiency oftransformation by homologous recombination is increased when the plasmidis cut by restriction enzyme digestion within the targeting DNA sequencehomologous in part to the target r-DNA sequence.

[0059] The host yeast cells are grown under conditions such that onlythose yeast cells that have been stably transformed, i.e., have had theplasmid and selectable marker stably integrated in the host cell byhomologous recombination will be able to grow. In a correctly targetedevent, the entire plasmid is stably incorporated contained in the hostyeast cell by homologous recombination of the targeting DNA sequence ofthe plasmid and the homologous target r-DNA clone sequence. Only thosefew host yeast cells that contain the desired, target r-DNA clonesequence (and have thereby undergone homologous recombination with thetargeting plasmid) are able to grow under the new growth conditions, dueto the introduction of the yeast-selectable marker gene contained on thetargeting plasmid.

[0060] The vast majority of the population of the host yeast cellscontaining r-DNA clone sequences that are not homologous to thetargeting DNA sequence contained on the plasmid, do not have the plasmidincorporated by homologous recombination and, therefore, do not acquirethe marker gene that is essential for growth under the selectionconditions. Therefore, it is preferable that any yeast-selectable markergene that is contained on the incoming targeting plasmid has beendeleted entirely or almost entirely from the genome of the host yeaststrain that is used for the vector. This prevents any spurioushomologous recombination events between the incoming yeast-selectablemarker gene and any other natural yeast genetic loci. If ayeast-selectable marker gene on the incoming targeting plasmid is notdeleted from the yeast genome, but is retained as a mutated,non-functional portion of the yeast chromosome, more positive scores forhomologous recombination will have to be screened to ensure that thehomologous recombination event has taken place between the targeting DNAsequence on the bacterial plasmid and the desired, target r-DNA clonesequence. Cells with the integrated marker can grow into colonies whenplated on appropriate selective media.

[0061] Alternatively, a yeast-selectable marker gene on the incomingtargeting DNA molecule can be a bacterial gene that confers drugresistance to yeast cells, e.g., the CAT or neo genes from Tn9 andTn903, or bacterial amino acid or amino acid nucleoside prototrophygenes, e.g., the E. coli argH, trpC, and pyrF genes.

[0062] Methods for plasmid purification, restriction enzyme digestion ofplasmid DNA and gel electrophoresis, use of DNA modifying enzymes,ligation, transformation of bacteria, transformation of yeast by thelithium acetate method, preparation and Southern blot analysis of yeastDNA, tetrad analysis of yeast, preparation of liquid and solid media forthe growth of E. coli and yeast, and all standard molecular biologicaland microbiological techniques can be carried out essentially asdescribed in Ausubel et al. (Ausubel, F. M. et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Interscience,New York, 1987).

[0063] Once the proper assembly of the invention chimeric scFv-INT-1protein and adhesion to the epitopes in target cell lines, e.g., tumorcell lines, has been tested in a non-pathogenic yeast cell (e.g.,Saccharomyces cerevisiae) homologous recombination can be used to inserta polynucleotide sequence encoding the chimeric scFv-INT1 into Candidaalbicans, and similar ex vivo experiments as those performed for S.cerevisiae will be performed to assure that replacement of the I domaindoes not seriously impair the proper folding and targeting of scFv-INT1.At this point, ex vivo experiments verifying adhesion of this mutant C.albicans strain to cancer cells are performed, in addition topreliminary in vivo mice experiments to ascertain that this targetingalone is adequate in mice to restrict pathogenicity and targeting totumors.

[0064] The most common model for human cancers is a murine subject thathas been transfected with human carcinomas. After an incubation periodvarying from weeks to months after carcinoma introduction to allowgrowth of test tumors, transfected mice will be treated with thegenetically modified C. albicans. Survival of the mice and tumorspreading are monitored over time. Biopsies of the tumorous tissues canalso be taken to investigate C. albicans invasion. By using large groupsof genetically identical mice, aggregate data can be collected.

[0065] Evolution has optimized certain organisms to invade mammaliantissue. The present invention harnesses this powerful and highlypathogenic trait to generate a new weapon against cancer and otherdiseases characterized by the presence of cells with adisease-associated antigen. In contrast to more indirect methodologiespreviously applied that recruit the natural immune system responses,fusion scFv-INT1 proteins targeted to disease-associated tissues willdirect aggressive invasion of the naturally invasive pathogen todiseased host tissue. The method of the present invention is a novelapproach to cancer treatment that recruits the previously untappedresource of pathogenic organisms (e.g. fungi) as potent and specifictherapy to eliminate diseased tissue characterized by disease-associatedantigens.

[0066] The invention will now be described by reference to the followingnon-limiting illustrative example:

EXAMPLE 1

[0067] Construction of the scFv-INT1 Fusion Gene

[0068] Using bulk genomic DNA from C. albicans, primers 1 and 2 (shownin FIGS. 2A-B) (SEQ ID NOS:2 and 3) are used for PCR amplification ofthe INT1 gene (available from GenBank under accession number U35070)(SEQ ID NO:1) as previously described (Gale 1996). These primers insertSacI and ApaI restriction sites at the 5′ and 3′ ends of the codingregion of INT1, respectively. These restriction sites are bothnonexistent in the ORF of the gene (see gene sequence in FIGS. 1A-W).The 5 kB product of this PCR reaction is isolated using a standardQiagen desalting kit, digested with the appropriate enzymes SacI andApaI, and ligated into predigested and dephosphorylated pBluescript IISK (+) phagemid plasmid according to the manufacturer's instructions(Product #212205, Stratagene, LaJolla, Calif.). ssDNA incorporating theINT1 gene is then generated using standard techniques with helper phageand uridine in dut⁻ ung⁻ strains of E. Coli according to themanufacturer's instructions.

[0069] To introduce multiple cloning sites in the ssDNA PCR product inthe place of the I domain of INT1, primer 7 (shown in FIGS. 2A-B) (SEQID NO:8) is used in a standard polymerase/ligase reaction; also thuseliminating the I domain. Isolation of the generated plasmids isperformed using standard techniques.

[0070] Single chain antibodies (scFvs) having the target antigen bindingregion of a desired antigen are generated using reverse transcriptasePCR of the bulk RNA from antibody-generating cell lines using primers 4to 6 (shown in FIGS. 2A-B) (SEQ ID NOS:5, 6, and 7). The binding regionsare subcloned into the cut and dephosphorylated plasmid prepared asdescribed above, and then a fusion gene is isolated and characterizedusing techniques described in Z. Eshhar et al., Methods in Enzymology8:133-142 (1995), except that Primers 4 to 6 differ from those shown inEshhar by including different restriction endonuclease sites, as INT1has restriction sites for the nucleases used by Eshhar. The primers usedto remove the binding regions of the heavy and light chains incorporatea linker that allows the now-assembled scFv-INT1 protein to have thebinding region activated and folded properly.

[0071] The chimeric INT1-scFv fusion protein is directly expressed in E.Coli for in vitro studies of folding and binding using known techniquesdescribed in Sections 10.0.1 and 16.1 to 16.7 of Current Protocols inMolecular Biology, Collected Volumes 1 to 4, edited by Ausubel, F. M. etal., John Wiley & Sons, 2000. In addition, the chimeric INT1-scFv fusionprotein is incorporated into S. Cerevisiae using an expression plasmidcontaining the nucleotide sequence that encodes the fusion protein forcell-cell studies and is incorporated back into Candida albicans byhomologous recombination using techniques described in Section 13.10.3of Current Protocols in Molecular Biology, supra. Thus, a tumor-specificorganism is readily accomplished.

[0072] It will be apparent to those skilled in the art that variouschanges may be made in the invention without departing from the spiritand scope thereof, and therefore, the invention encompasses embodimentsin addition to those specifically disclosed in the specification, butonly as indicated in the appended claims.

What is claimed is:
 1. A chimeric pathogenic organism wherein virulenttransformation of the organism is controlled in the wild-type organismby binding of the I domain of a surface integrin-like protein to a cell,said chimeric organism comprising a chimeric surface integrin-likefusion protein in which the I domain is replaced by an antibody fragmentthat binds a disease-associated antigen on a cell, wherein binding ofthe antibody fragment to the disease-associated antigen triggersvirulent transformation of the chimeric pathogenic organism so as tocause the organism to infiltrate the cell.
 2. The chimeric pathogenicorganism of claim 1, wherein the antibody fragment is a single chainantibody.
 3. The chimeric pathogenic organism of claim 1, wherein theantibody fragment binds to an antigen on a tumor cell.
 4. The chimericpathogenic organism of claim 3, wherein the antigen is contained in anabnormal surface protein of the tumor cell.
 5. The chimeric pathogenicorganism of claim 3, wherein the antigen is selected from the groupconsisting of GAG-72, ERBB2, EGP-2, CEA, CD44, I-FR, neu, and the Lewis(Y) tumor associated antigen.
 6. The chimeric pathogenic organism ofclaim 3, wherein the tumor cell is selected from the group consisting ofadenocarcinoma of colon, ovary or breast; cervical nonmucinous ovariancarcinoma; and breast, ovarian, colorectal, and pancreatic cancers.
 7. Achimeric pathogenic C. albicans comprising: an integrin1 (INT1) fusionprotein in which the I domain is replaced by an antibody fragment thatbinds to a disease-associated antigen on a diseased cell, a disabledwild-type high affininity iron transporter (CAFTR) gene, and a DNAconstruct comprising a wild-type CAFTR gene under the control of anenhanced filamentous growth protein (EFG1p) response element, whereinbinding of the antibody to the disease-associated antigen triggersexpression of the CAFTR gene in the DNA construct and filamentoustransformation in the chimeric pathogenic C albicans.
 8. The chimericpathogenic C. Albicans of claim 7, wherein the antibody fragment is asingle chain antibody.
 9. The chimeric pathogenic C. Albicans of claim7, wherein the antibody fragment binds to an antigen on a tumor cell.10. The chimeric pathogenic C. Albicans of claim 9, wherein the antigenis contained in an abnormal surface protein of the tumor cell.
 11. Thechimeric pathogenic C. Albicans of claim 9, wherein the antigen isselected from the group consisting of GAG-72, ERBB2, EGP-2, CEA, CD44,I-FR, neu, and the Lewis (Y) tumor associated antigen.
 12. The chimericpathogenic C. Albicans of claim 9, wherein the tumor cell is selectedfrom the group consisting of adenocarcinoma of colon, ovary or breast;cervical nonmucinous ovarian carcinoma; and breast, ovarian, colorectal,and pancreatic cancers.
 13. A method for treating a disease associatedwith the presence of cells having a disease-associated surface antigenin a subject in need thereof, said method comprising: administering tothe subject a therapeutically effective amount of a chimeric pathogenicorganism according to claim 1 so as to cause binding of the antibodyfragment to the disease-associated antigen on the cells, therebytreating the disease by triggering infiltration of the chimericpathogenic organism into the cells without substantial damage to healthycells.
 14. The method of claim 13, wherein the antibody fragment is asingle chain antibody.
 15. The method of claim 13, wherein the antibodyfragment binds to an antigen on a tumor cell.
 16. The method of claim15, wherein the disease-associated antigen is contained in an abnormalsurface protein of the tumor cell.
 17. The method of claim 16, whereinthe antigen is selected from the group consisting of GAG-72, ERBB2,EGP-2, CEA, CD44, I-FR, neu, and the Lewis (Y) tumor associated antigen.18. The method of claim 15, wherein the tumor cell is selected from thegroup consisting of adenocarcinoma of colon, ovary or breast; cervicalnonmucinous ovarian carcinoma; and breast, ovarian, colorectal, andpancreatic cancers.
 19. The method of claim 15, wherein the antigen is atumor marker.
 20. The method of claim 19, wherein the method furthercomprises administering to the subject a therapeutic amount of animmunosuppressive agent.
 21. The method of claim 20, wherein theimmunosuppressive agent is selected from the group consisting ofcyclosporin A, OKT3, FK506, mycophenolate mofetil (MMF), azathioprine, acorticosteroid, an antilymphocyte globulin, and an antithymocyteglobulin.
 22. A method for generating a chimeric therapeutic organismfrom a pathogenic organism that possesses in the wild-type anintegrin-like protein with an I domain, said method comprising:replacing the I domain in the integrin-like protein of the pathogenicorganism with an antibody fragment that binds to a disease-associatedantigen on a diseased cell; wherein the wild-type pathogenic organismundergoes virulent transformation by binding of the I domain of thesurface integrin-like protein to a cell, and wherein the chimerictherapeutic organism undergoes virulent transformation by binding of theantibody fragment to the disease-associated antigen on the cell.
 23. Themethod of claim 22, wherein the pathogenic organism is C. albicans andwherein the method further comprises disabling the wild-type CAFTR genein the C. albicans, and introducing a DNA construct comprising awild-type CAFTR gene under the control of a EFG1p response element,wherein binding of the antibody fragment to the disease-associatedantigen triggers expression of the CAFTR gene in the DNA construct andfilamentous transformation in the chimeric pathogenic C. albicans. 24.The method of claim 23, wherein the antibody fragment is a single chainantibody.
 25. The method of claim 23, wherein the antibody fragmentbinds to an antigen on a tumor cell.
 26. The method of claim 25, whereinthe disease-associated antigen is contained in an abnormal surfaceprotein of the tumor cell.